The retroviruses we study were isolated from wild mice and cause a non- inflammatory neurodegenerative disease in mice similar to that caused by the unconventional agents such as scrapie. We are using this animal model to study the pathogenesis of retrovirus-induced neurodegeneration. Implant studies: The mechanism of neurodegeneration appears to be indirect since the neurons which degenerate appear not to be infected. We showed that microglial cells are a important target of virus infection and that infected microglial cells colocalized with the neuronal cytopathology. We have now shown that microglial cells isolated from the brains of neonatal mice, infected in vitro and implanted into the brains of 10 day old can induce local spongiform neuropathology. The cells migrate to specific sites within the brain remote from the sites of injection and lesions have been detected in the thalamus, colliculus and along the corpus callosum. This is the first direct demonstration that microglial cells infected with a retrovirus may be neurotoxic in vivo, a finding which has direct implications to understanding HIV encephalopathy. Envelope protein: The viral envelope protein contains the determinants of neurovirulence, but the nature of this effect is not understood. We used two related retroviruses, both of which infect the brain at high levels, but only one of which is neurovirulent. The viral envelope protein was examined in microglial cells infected in vitro with each virus. Interestingly, microglial cells infected with the neurovirulent virus expressed only the precursor of the envelope protein, suggesting that proteolytic cleavage of this protein did not occur in these cells. In contrast, the env precursor was proteolytically cleaved in cells infected with the non-neurovirulent virus. Since both of these viruses infect a comparable spectrum of cell types in the brain, this suggests that proteolytic processing may be proximally involved in disease pathogenesis. In vitro model of neurodegeneration: We are continuing our attempts to develop an organotypic slice culture system which will recapitulate the neuropathology seen in vivo. Infected slice cultures derived from the brain stem and the spinal cord have been maintained for weeks in culture. The retrovirus infection is persistent and the infected cells have been identified by dual color immunofluorescence using cell-type specific markers. The major infected cell in spinal cord cultures is the microglial cell. The viral protein profile expressed in these cultures has also been followed as a function of time after infection. As seen in vivo there are several isoforms of envelope protein whereas the precursor of the core (gag) proteins is expressed as a single isoform. Thus, both the target cells of the infection in vitro and the processing of the viral proteins appears to mimic that seen in mice inoculated with this virus.